Project Abstract A novel, biologically inspired strategy will be used to improve DMSO-free preservation of cardiomyocytes derived from human induced pluripotent stem (hiPS) cells cultured as aggregates. Multicellular systems respond poorly to conventional preservation methods. In the previous funding cycle, a differential evolution algorithm was used to optimize the preservation of cells using combinations of sugars, sugar alcohols and amino acids. This approach mimicked the strategies of plants and other simple organisms that survive environmental stresses. Studies characterizing the behavior of multicomponent osmolyte solutions demonstrated that osmolytes present in these solutions interact with each other to modify the behavior of water during freezing which manifests itself at a molecular level (hydrogen bonding) as well as changes in microstructure. The osmolytes also interact with critical biological structures in the cell to stabilize them. Low temperature Raman spectroscopy studies demonstrated that multicellular systems are sensitive to undercooling (the temperature difference between the freezing temperature and the temperature at which ice forms in the extracellular solution). As we transition from single cells to aggregates, once again, we are looking to nature for inspiration. The wood frog uses combinations of osmolytes, suppressed cell metabolism, and minimizes undercooling as it adapts to freezing conditions in the winter. We are proposing a similar approach to improving preservation of cardiomyocyte aggregates. The studies described in the application characterize the freezing response of iPSC-derived cardiomyocytes at the committed cardiac progenitor stage and when formed into fully differentiated, multicellular cardiac constructs. The influence of undercooling on post thaw recovery of fully differentiated multicellular cardiac constructs will also be determined. The influence of suppressing cell metabolism on post thaw recovery of multicellular constructs will also be quantified. On a fundamental level, these studies will advance our understanding of the freezing behavior of more complex multicellular systems. On an applied level, the proposed investigation has the potential to preservation of cells differentiated from hiPS cells through the use of naturally inspired strategies.